Andrew C. Penn

Molecular determinants of AMPA receptor subunit assembly

AMPA-type (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) glutamate receptors (AMPARs) mediate post-synaptic depolarization and fast excitatory transmission in the central nervous system. AMPARs are tetrameric ion channels that assemble in the endoplasmic reticulum (ER) in a poorly understood process. The subunit composition determines channel conductance properties and gating kinetics, and also regulates vesicular traffic to and from synaptic sites, and is thus critical for synaptic function and plasticity. The distribution of functionally different AMPARs varies within and between neuronal circuits, and even within individual neurons. In addition, synapses employ channels with specific subunit stoichiometries, depending on the type of input and the frequency of stimulation. Taken together, it appears that assembly is not simply a stochastic process. Recently, progress has been made in understanding the molecular mechanisms underlying subunit assembly and receptor biogenesis in the ER. These processes ultimately determine the size and shape of the postsynaptic response, and are the subject of this review.

The concentrations of calcium buffering proteins in mammalian cochlear hair cells.

Calcium buffers are important for shaping and localizing cytoplasmic Ca2+ transients in neurons. We measured the concentrations of the four main calcium-buffering proteins (calbindin-D28k, calretinin, parvalbumin-α, and parvalbumin-β) in rat cochlear hair cells in which Ca2+ signaling is a central element of fast transduction and synaptic transmission. The proteins were quantified by calibrating immunogold tissue counts against gels containing known amounts of each protein, and the method was verified by application to Purkinje cells in which independent estimates exist for some of the protein concentrations. The results showed that, in animals with fully developed hearing, inner hair cells had \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\frac{1}{10}\) \end{document} of the proteinaceous calcium buffer of outer hair cells in which the cell body contained parvalbumin-β (oncomodulin) and calbindin-D28k at levels equivalent to 5 mm calcium-binding sites. Both proteins were partially excluded from the hair bundles, which may permit fast unbuffered Ca2+ regulation of the mechanotransducer channels. The sum of the calcium buffer concentrations decreased in inner hair cells and increased in outer hair cells as the cells developed their adult properties during cochlear maturation. The results suggest that Ca2+ has distinct roles in the two types of hair cell, reflecting their different functions in auditory transduction. Ca2+ is used in inner hair cells primarily for fast phase-locked synaptic transmission, whereas Ca2+ may be involved in regulating the motor capability underlying cochlear amplification of the outer hair cell. The high concentration of calcium buffer in outer hair cells, similar only to skeletal muscle, may protect against deleterious consequences of Ca2+ loading after acoustic overstimulation.

Gating motions underlie AMPA receptor secretion from the endoplasmic reticulum

Ion channel biogenesis involves an intricate interplay between subunit folding and assembly. Channel stoichiometries vary and give rise to diverse functions, which impacts on neuronal signalling. AMPA glutamate receptor (AMPAR) assembly is modulated by RNA processing. Here, we provide mechanistic insight into this process. First, we show that a single alternatively spliced residue within the ligand‐binding domain alters AMPAR secretion from the ER. Local contacts differ between Leu758 of the GluR2‐flop splice form as compared with the flip‐specific Val758, which is transmitted globally to alter resensitization kinetics. Detailed biochemical and functional analysis of mutants suggest that AMPARs sample the gating cascade prior to ER export. Irreversibly locking the receptor within various states of the cascade severely attenuates ER transit. Alternative RNA processing by contrast, shifts equilibria between transition states reversibly and thereby modulates secretion kinetics. These data reveal how RNA processing tunes AMPAR biogenesis, and imply that gating transitions in the ER determine iGluR secretory traffic.

Subunit-selective N-terminal domain associations organize the formation of AMPA receptor heteromers

The assembly of AMPA‐type glutamate receptors (AMPARs) into distinct ion channel tetramers ultimately governs the nature of information transfer at excitatory synapses. How cells regulate the formation of diverse homo‐ and heteromeric AMPARs is unknown. Using a sensitive biophysical approach, we show that the extracellular, membrane‐distal AMPAR N‐terminal domains (NTDs) orchestrate selective routes of heteromeric assembly via a surprisingly wide spectrum of subunit‐specific association affinities. Heteromerization is dominant, occurs at the level of the dimer, and results in a preferential incorporation of the functionally critical GluA2 subunit. Using a combination of structure‐guided mutagenesis and electrophysiology, we further map evolutionarily variable hotspots in the NTD dimer interface, which modulate heteromerization capacity. This ‘flexibility’ of the NTD not only explains why heteromers predominate but also how GluA2‐lacking, Ca2+‐permeable homomers could form, which are induced under specific physiological and pathological conditions. Our findings reveal that distinct NTD properties set the stage for the biogenesis of functionally diverse pools of homo‐ and heteromeric AMPAR tetramers.

Glutamate-Induced AMPA Receptor Desensitization Increases Their Mobility and Modulates Short-Term Plasticity through Unbinding from Stargazin

Short-term plasticity of AMPAR currents during high-frequency stimulation depends not only on presynaptic transmitter release and postsynaptic AMPAR recovery from desensitization, but also on fast AMPAR diffusion. How AMPAR diffusion within the synapse regulates synaptic transmission on the millisecond scale remains mysterious. Using single-molecule tracking, we found that, upon glutamate binding, synaptic AMPAR diffuse faster. Using AMPAR stabilized in different conformational states by point mutations and pharmacology, we show that desensitized receptors bind less stargazin and are less stabilized at the synapse than receptors in opened or closed-resting states. AMPAR mobility-mediated regulation of short-term plasticity is abrogated when the glutamate-dependent loss in AMPAR-stargazin interaction is prevented. We propose that transition from the activated to the desensitized state leads to partial loss in AMPAR-stargazin interaction that increases AMPAR mobility and allows faster recovery from desensitization-mediated synaptic depression, without affecting the overall nano-organization of AMPAR in synapses.

Activity-regulated RNA editing in select neuronal subfields in hippocampus.

RNA editing by adensosine deaminases is a widespread mechanism to alter genetic information in metazoa. In addition to modifications in non-coding regions, editing contributes to diversification of protein function, in analogy to alternative splicing. However, although splicing programs respond to external signals, facilitating fine tuning and homeostasis of cellular functions, a similar regulation has not been described for RNA editing. Here, we show that the AMPA receptor R/G editing site is dynamically regulated in the hippocampus in response to activity. These changes are bi-directional, reversible and correlate with levels of the editase Adar2. This regulation is observed in the CA1 hippocampal subfield but not in CA3 and is thus subfield/celltype-specific. Moreover, alternative splicing of the flip/flop cassette downstream of the R/G site is closely linked to the editing state, which is regulated by Ca2+. Our data show that A-to-I RNA editing has the capacity to tune protein function in response to external stimuli.

Hippocampal LTP and contextual learning require surface diffusion of AMPA receptors

Long-term potentiation (LTP) of excitatory synaptic transmission has long been considered a cellular correlate for learning and memory. Early LTP (less than 1 h) had initially been explained either by presynaptic increases in glutamate release or by direct modification of postsynaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor function. Compelling models have more recently proposed that synaptic potentiation can occur by the recruitment of additional postsynaptic AMPA receptors (AMPARs), sourced either from an intracellular reserve pool by exocytosis or from nearby extra-synaptic receptors pre-existing on the neuronal surface. However, the exact mechanism through which synapses can rapidly recruit new AMPARs during early LTP remains unknown. In particular, direct evidence for a pivotal role of AMPAR surface diffusion as a trafficking mechanism in synaptic plasticity is still lacking. Here, using AMPAR immobilization approaches, we show that interfering with AMPAR surface diffusion markedly impairs synaptic potentiation of Schaffer collaterals and commissural inputs to the CA1 area of the mouse hippocampus in cultured slices, acute slices and in vivo. Our data also identify distinct contributions of various AMPAR trafficking routes to the temporal profile of synaptic potentiation. In addition, AMPAR immobilization in vivo in the dorsal hippocampus inhibited fear conditioning, indicating that AMPAR diffusion is important for the early phase of contextual learning. Therefore, our results provide a direct demonstration that the recruitment of new receptors to synapses by surface diffusion is a critical mechanism for the expression of LTP and hippocampal learning. Since AMPAR surface diffusion is dictated by weak Brownian forces that are readily perturbed by protein–protein interactions, we anticipate that this fundamental trafficking mechanism will be a key target for modulating synaptic potentiation and learning.

Activity-Mediated AMPA Receptor Remodeling, Driven by Alternative Splicing in the Ligand-Binding Domain

Summary The AMPA-type glutamate receptor (AMPAR) subunit composition shapes synaptic transmission and varies throughout development and in response to different input patterns. Here, we show that chronic activity deprivation gives rise to synaptic AMPAR responses with enhanced fidelity. Extrasynaptic AMPARs exhibited changes in kinetics and pharmacology associated with splicing of the alternative flip/flop exons. AMPAR mRNA indeed exhibited reprogramming of the flip/flop exons for GluA1 and GluA2 subunits in response to activity, selectively in the CA1 subfield. However, the functional changes did not directly correlate with the mRNA expression profiles but result from altered assembly of GluA1/GluA2 subunit splice variants, uncovering an additional regulatory role for flip/flop splicing in excitatory signaling. Our results suggest that activity-dependent AMPAR remodeling underlies changes in short-term synaptic plasticity and provides a mechanism for neuronal homeostasis.

AMPA Receptor Assembly: Atomic Determinants and Built-In Modulators

Glutamate-gated ion channels (iGluRs) predominantly operate as heterotetramers to mediate excitatory neurotransmission at glutamatergic synapses. The subunit composition of the receptors determines their targeting to synaptic sites and signalling properties and is therefore a fundamental parameter for neuronal computations. iGluRs assemble as obligatory or preferential heteromers; the mechanisms underlying this selective assembly are only starting to emerge. Here we review recent work in the field and provide an in-depth update on atomic determinants in the assembly domains, which have been facilitated by recent advances in iGluR structural biology. We also discuss the role of alternative RNA processing in the ligand-binding domain, which modulates a central subunit interface and has the capacity to modulate receptor formation in response to external cues. Finally, we review the emerging physiological significance of signalling via distinct iGluR heterotetramers and provide examples of how recruitment of functionally diverse receptors modulates excitatory neurotransmission under physiological and pathological conditions.

miR-92a regulates expression of synaptic GluA1-containing AMPA receptors during homeostatic scaling

We investigated whether microRNAs could regulate AMPA receptor expression during activity blockade. miR-92a strongly repressed the translation of GluA1 receptors by binding the 3′ untranslated region of rat GluA1 (also known as Gria1) mRNA and was downregulated in rat hippocampal neurons after treatment with tetrodotoxin and AP5. Deleting the seed region in GluA1 or overexpressing miR-92a blocked homeostatic scaling, indicating that miR-92a regulates the translation and synaptic incorporation of new GluA1-containing AMPA receptors.